Native chemical ligation (NCL) is a chemo-selective reaction that occurs at physiological pH between an N-terminal cysteine residue and a C-terminal peptide thioester (Dawson et al., Science 1994, 266, 776-779; Dawson et al., Annu. Rev. Biochem. 2000, 69, 923-960; Yeo et al., Chem.-Eur. J. 2004, 10, 4664-4672). In the first step of ligation, a reversible trans-thioesterification takes place between the C-terminal thioester and the sulfhydryl group from the N-terminal cysteine residue. The ligated peptide thioester then undergoes a rapid, irreversible and spontaneous intramolecular S→N shift, generating the thermodynamically favored native amide bond at the ligation junction. NCL occurs uniquely at an N-terminal cysteine residue regardless of the presence of any additional internal cysteine residues and, as this ligation method is compatible with both carbohydrates and peptides, provides access to glycopeptides.
The applicability of NCL is reduced when peptide segments are poorly soluble in aqueous buffer. Since NCL is usually performed in aqueous buffers, this can present complications when one of the reactants to be ligated has hydrophobic character. Recently, some researchers have attempted to use native chemical ligation to link selected reactants to membrane-spanning domain fragments of transmembrane proteins. Otaka et al. covalently linked two membrane-embedded transmembrane peptide domains at a ligation site that was situated in the hydrophilic extracellular loop region (Chem Commun., 2004, 1722-1723). Hunter et al. attached a small soluble peptide to the end of a transmembrane peptide embedded in a cubic lipidic phase matrix (Bioconjugate Chem., 2004, 15:437; U.S. Pat. Publ. 20030018169, published Jan. 23, 2003).
There remains, however, a need for reliable processes for chemical ligation of a wide variety of hydrophobic molecules including compounds that contain lipid and/or carbohydrate moieties.